High level expression of recombinant proteins produced in bacteria, such as E. coli, often results in formation of insoluble aggregates within the bacterial cell, known as inclusion bodies (Shein et al., Bio/Technology 7:1141-49, 1989). An inclusion body protein is one that is, in general, overexpressed in the host, which at later stages of expression or purification is visible by phase contrast microscopy as a precipitate. Inclusion bodies are electron-dense amorphous particles which have a discrete border to the cytoplasm but are not surrounded by a membrane (Schoemaker et al., EMBO J. 4:775-780, 1985). During the preparation of inclusion bodies, various types of interactions may lead to secondary adsorption of other contaminants such as, endotoxins, cell wall debris, and lipids (Marston FAO, Biochem. J. 240:1-12, 1986). The average particle size of inclusion bodies is dependent on the particular target protein expressed, the host strain, the expression system and the culture medium used and may be in the range from 0.07 μm for human growth hormone (Blum P et al., Bio/Technology 10: 30)-304, 1992) to 1.5 μm for β-lactamase (Bowden et al., Bio/Technology 9: 725-730, 1991). A further description of inclusion body can be found in U.S. Pat. No. 4,512,922, which refers to inclusion bodies as “refractile bodies.”
Inclusion bodies are generally harvested from cell lysate through several centrifugation and wash steps after the cells are lysed (e.g., by lysis by lysozyme, ultrasound treatment or high pressure homogenization). See, for example, U.S. Pat. Nos. 4,511,503; 4,518,526; 5,605,691; and 6,936,699. The purified inclusion bodies are then dissolved or denatured, with, e.g., a detergent or other solution (urea. SDS, guanidine hydrochloride), which causes the insoluble protein molecules to unfold and become soluble. The denaturant may subsequently removed, for example, by dialysis, by molecular sieve, or by centrifugation at high speed to remove higher molecular weight components and decant the denaturant. The recombinant protein is then isolated and refolded to form correct high order structures which are biologically active.
In order to insure the most efficient refolding reaction, it is important to control the amount and concentration of the proteins in the refolding reaction. The protein recovered from the inclusion bodies is typically determined by high performance liquid chromatography (HPLC) analyses of an aliquot of the inclusion body harvest. However, real-time analysis by HPLC methods are complex and time-consuming nature of the process.
Thus, there remains a need in the art for more efficient and accurate methods of determining recombinant protein levels produced during recombinant protein production.